JPH04293537A - Apparatus and method for preparing liposome - Google Patents

Abstract

PURPOSE:To obtain a liposome preparing apparatus capable of enhancing the efficiency and scale of the emulsification-gel formation-volatic swing-vesicle formation process in an inverse phase evaporation method. CONSTITUTION:A liposome preparing apparatus is constituted of a sample container 3 equipped with the temp. adjusting jacket 2 connected to the ultrasonic converter of an ultrasonic transmitter, the chamber 4 covering said container 3, a sample injection and discharge mechanism 5, a dilution water injection mechanism 6 and an aspirator part 7 for evacuating the chamber 4 and can perform an emulsification-gel formation-volatic swing-vesicle formation process in an inverse phase evaporation method in the sample container 3 as a single container whithin a short time.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liposome preparation device that can easily prepare liposomes based on a reverse phase evaporation method, and a liposome preparation method using the device. For example, the present invention relates to an enzyme-encapsulated liposome preparation device used to accelerate the ripening of cheese and a liposome preparation method using the device.

0002

[Prior Art] Liposomes are endoplasmic reticulum with a closed bilayer membrane formed when phospholipids are suspended in an aqueous solution. Large unilamellar liposomes (
(LUV) etc. Since this closed bilayer membrane is similar to biological membranes, research on its use in the field of medicine is being actively conducted. There are a wide variety of conventional liposome preparation methods, and depending on the method, the shape of the liposome,
The size and retention efficiency of the substance sealed inside also vary. The main preparation methods include portake swing method (forming liposomes-MLV), ultrasound method (SUV), cholic acid removal method (SUV), freeze-thaw fusion method (LUV), and reverse phase evaporation method (REV-LUV). ) etc. Reverse-phase evaporation has the highest retention efficiency for encapsulated substances, which is said to be around 60% for low molecular weight substances. In any case, after preparation, dialysis,
Use gel filtration, UF membrane, etc. to remove unencapsulated substances. Currently commercially available liposome preparation devices include Liposomat (DIA-CHEMA, Germany) and Microfluidizer system (Microfluid, USA).
ics), Extruder (Canada, Lipex)
Biomembrane Inc.) etc.

0003

Problems to be Solved by the Invention As mentioned above, the reverse phase evaporation method is said to have the highest retention efficiency of substances among the various conventional preparation methods. A typical REV preparation operation using the reverse phase evaporation method is as follows. In other words, lipids. The drug mixture is placed in a test tube or the like and irradiated with ultrasonic waves to form a W/O emulsion. Thereafter, the mixture is transferred to an eggplant-shaped flask, and the solvent is distilled off under reduced pressure using an evaporator to form a gel. Once removed from the evaporator, it is vibrated with a touch mixer or the like to form a lipid bilayer membrane in which hydrophobic groups are bonded together. This time series is W/O/W. The sample is again set in the evaporator, and while the sample is being spread into a thin film, the remaining solvent is distilled off under reduced pressure to form a vesicle. After this, dilute with distilled water or buffer, remove unencapsulated material, and REV.
shall be. In addition, the system during this time is W (water) O (oil) mixture → W
Since it changes from /O to W/0/W, it is necessary to confirm this state by measuring conductivity, etc. The conventional method described above has problems in terms of efficiency, such as complicated and time-consuming operations because it is carried out using an eggplant-shaped flask. The most complicated steps in the reverse phase evaporation method are emulsification-gel formation-voltage swing-vesicle formation.

However, although it is said that stabilization of liposomes and establishment of a large-scale preparation method are two conditions for the practical use of liposomes, the above-mentioned methods that seem to lead to increased efficiency in liposome preparation At present, no liposome preparation device has been announced based on the principle of the reversed phase evaporation method, which can carry out complicated steps in a single hour and can be scaled up. Therefore, the present invention utilizes the reverse phase evaporation method for emulsification-gel formation-voltage swing-Vesi
The object of the present invention is to provide a liposome preparation device that can streamline and scale up the cle formation process, and a liposome preparation method using the device.

[0005]

[Means for Solving the Problems] In order to achieve the above objects, the present invention provides the following liposome preparation device and a liposome preparation method using the device. That is, a sample container that also serves as the horn of the ultrasonic converter, a chamber that covers the sample container, a sample injection/discharge mechanism that supplies the sample to the sample container and collects REV, and a dilution water injection mechanism that supplies dilution water to the sample container. This is a liposome preparation device consisting of an aspirator section for reducing the pressure inside the chamber. However, the sample container described above is equipped with a temperature adjustment jacket connected to a converter, and the sample injection/discharge mechanism is connected to the tip of the piping that communicates with the sample tank and the REV tank via a pump and a switching valve. It consists of something facing the sample container. or,
The dilution water injection mechanism consists of a pipe with a valve and pump connected to the buffer tank, with the tip facing the sample container, and the aspirator is connected to the upper part of the chamber through a mesh. . Furthermore, a method for preparing liposomes using the above-mentioned apparatus is a method for preparing lipids. This liposome preparation method comprises supplying a drug mixture to a sample container in the liposome preparation device described above, allowing emulsion-vesicle formation to occur in the same sample container, and then diluting with a diluent and collecting REV from the sample container. .

[0006]

[Function] This liposome preparation device and method are capable of carrying out the steps of emulsification, gel formation, voltage swing, and vesicle formation by reverse phase evaporation in a short time in a single sample container.

[0007]

EXAMPLES The liposome preparation device of the present invention and its method of use are shown in FIGS. 1 and 2, and its preparation flow is shown in FIG. According to the flow eggPC, Chol, DCP
is dissolved in dichloromethane, the solvent is removed with an evaporator to obtain a lipid mixture, this is dispersed in diethyl ether, and an enzyme solution (phosphate buffer) is further added and mixed. It has been shown that liposomes can be prepared by injecting And in the liposome preparation device, emulsification-Ve by reverse phase evaporation method is used.
Sicle formation is combined into a single vessel operation, and the lipid. The process involves forming vesicles all at once by irradiating the drug mixture with ultrasound under reduced pressure, and then diluting this with phosphate buffer to form REV. In addition, in the above flow, organic solvents for lipid dissolution include organic solvents commonly used for liposome preparation such as chloroform and hexane instead of diethyl ether, and commonly used organic solvents such as soybean lecithin and various synthetic phospholipids instead of eggPC. Any lipid used may be used.

Next, based on FIG. 1, emulsification by reverse phase evaporation method -
A liposome preparation device for forming vesicles will be explained. That is, the liposome preparation device includes a sample container (3) in a temperature adjustment jacket (2) connected to a converter (1) of an ultrasonic transmitting device, a chamber (4) that covers this, a sample injection/discharge mechanism (5), and a dilution mechanism. It consists of a water injection mechanism (6) and an aspirator part (7) for reducing the pressure inside the chamber (4). Sample container (3)
is connected to the ultrasonic converter (1) with a temperature adjustment jacket (2) and also serves as a horn, and is made of an inverted conical cup and is placed on top of the jacket (2) to process the sample here. become. The chamber (4) covering the sample container (3) as described above is made of transparent acrylic and forms a depressurized space together with the sample container (3). A mesh (8) is placed in the middle of the chamber (4) to prevent the foamed sample from escaping to the aspirator side. (9) is a pipe connected to an aspirator (not shown) and opens at the upper part of the chamber (4) where the cooling water inlet/outlet (26) (27) is located.
) and sample container (3). Ultrasonic converter (
1) vibrates the sample container (3), which also serves as a horn, thereby performing emulsification, gel formation, and vesicle formation. Note that this part can also be replaced with another stirring type emulsifier integrated with the sample container (3). A conductivity measuring electrode (10) is provided in the chamber (4) to detect the phase inversion state of the sample.

The basic configuration of the liposome preparation apparatus of the present invention has been described above, and in order to achieve a continuous batch system, there is a sample injection/discharge mechanism (5) and a dilution water injection mechanism (6). The sample injection/discharge mechanism (5) is a device for injecting the sample into the sample container (3) and discharging the REV, and includes two three-way valves (
11) (12), a stop valve (14), and a micro pump (13). They are arranged in sequence, and the tips of the main pipes (15) are connected to a nozzle (19) facing the sample container (3). The three-way valve (11) is a valve for switching between the suction pipe (16), the air discharge pipe (17), and the main pipe (15) that communicate with the sample tank (20).
is a valve for switching between the discharge pipe (18) and the main pipe (15) communicating with the REV tank (21). The sample is injected from the sample tank (20) through the three-way valve (11) (12) and the stop valve (13), and is sucked into the sample container (3) by the micro pump (13) through the suction pipe (16) and from the nozzle (19). Inject into. After injection, the sample in the main pipe (15) is pushed out through the three-way valve (11) to the air discharge pipe (17). After this, the stop valve (14) is closed and the lipid in the sample container (3) is removed. Process the drug mixture sample. For processed samples, open the stop valve (14), switch the three-way valve (12) to the REV tank side, and pump the micropump (1).
3) Discharge.

Next, the dilution water injection mechanism (6) supplies the sample container from the buffer tank (22) through the pipe (23) and the stop valve (24) and from the nozzle (28) at the tip of the pipe (23) using the micro pump (25). (3).

The preparation operation of the liposome preparation device is as follows. In other words, lipids. The drug mixture is in the sample tank (20)
from the micro pump (1) through the three-way valve (11) (12).
3) into the sample container (3). Next, an aspirator (not shown) is operated to reduce the pressure inside the chamber (4), and the ultrasonic converter (1) irradiates ultrasonic waves to form a vesicle. After the solvent has been completely removed, the pressure inside the chamber (4) is returned to normal, and the buffer is injected from the buffer tank (22) into the sample container (3) using the dilution water injection mechanism (6), and the ultrasonic converter is injected into the sample container (3). (1) Lightly irradiate the vesicle with ultrasound to dilute it. Then, the micro pump (13) is reversely rotated, the three-way valve (12) is switched, and the sample is put into the REV tank (21) through the discharge pipe (18), completing one process. The basic processing method and mechanism of vesicle formation in the chamber (4) as described above are as follows. First, W/
From O emulsification to gel formation. Furthermore, by gradually reducing the pressure and irradiating pulses for several seconds every few minutes, W/O
Phase inversion to /W and vesicle formation proceed simultaneously. At this time, the gel-like sample foams and becomes a thin film, facilitating the removal of the solvent. The ultrasonic irradiation conditions are controlled by the lipids, their mixing ratio, and the amount prepared.

The apparatus of the present invention is as described above, but the ultrasonic irradiation part of the apparatus prototyped for experimental purposes was manufactured by Branson S.
Using a modified cup-shaped horn of onifier 250, cover it with an acrylic cylinder and connect it to the aspirator.
It was possible to reduce the pressure inside. The sample used for the experiment was
60 μmol of a lipid mixture consisting of eggPC:Chol:DCP=80:15:5 was dispersed in 3ml of diethyl ether, mixed with 1ml of phosphate buffer, and injected into a cup-shaped horn. The preparation was carried out under the following conditions. (1) Emulsification (W/O emulsion formation) Pulse irradiation for 15 seconds at an output of about 50 W under normal pressure. (2) Vesicle formation, solvent removal, vacuum degree approximately 400
Irradiate pulses with an output of about 25W in mmHg for a few seconds every 2 minutes,
It takes 15 minutes. (3) Solvent Removal The liposomes prepared using a prototype device at a vacuum level of 680 mmHg and a required time of 15 minutes or more had a good formation condition, and most particles with a particle size of 1 to 2 μm were observed. Some particles with a diameter of 1 μm or less were also observed, but their shape was unknown. Moreover, almost no ether odor was detected. This is thought to be because the solvent is removed relatively quickly because the sample becomes a foamy thin film due to the reduced pressure and ultrasonic irradiation. From the above results, we were convinced that liposomes could be easily prepared, and we completed the device and method of the present invention.

Next, the experimental examples according to the present invention will be further described as follows. (Experiment Example 1) Hydrogenated purified egg yolk lecithin (Asahi Kasei R-2
After dissolving 195 mg of phospholipid (95% or more), 17.5 mg of cholesterol, and 8 mg of dicetyl phosphate in 15 ml of dichloromethane, the solvent was removed using an evaporator to obtain a lipid mixture. Dissolve this in 15 ml of diethyl ether, and then add 1% enzyme solution (enzyme - Amano, Protease A, solvent - 3mM phosphate buffer p
After adding 5 ml of H7.2) and mixing gently, the mixture was injected into a liposome preparation device to prepare liposomes. The preparation conditions were to perform pulse irradiation at 45°C under normal pressure for 15 seconds at an output of 25 W, and after confirming that it became W/O with a conductivity meter, the pressure was gradually lowered to a vacuum level of 600 mmHg and a pulse of about 15 W was applied. Irradiation was carried out at a rate of several seconds every 2 minutes, and this was continued for 10 minutes. The temperature is 45°C
The temperature was gradually lowered from 20°C to 20°C. This liposome was diluted 50 times with 3mM phosphate buffer, and the UF module (Tosoh UF-LMF molecular weight cutoff 1 x 10
6) to remove unencapsulated enzyme. Next, the amount of enzyme encapsulated in the liposomes was determined by measuring enzyme activity. The enzyme retention efficiency is determined by the amount of Tr in the liposome dispersion.
The difference between the activity when the liposome membrane was destroyed by adding itonX-100 and the activity of the system without adding TritonX-100 was determined, and this was divided by the total activity before UF treatment. The retention efficiency was equivalent to Comparative Example 1 described below. Further, the preparation time in the liposome preparation device was about 15 minutes.

(Experimental Example 2) Egg yolk lecithin (Wako Pure Chemical Industries, Ltd.)
After dissolving 35 mg (for biochemistry), 3.5 mg of cholesterol, and 1.6 mg of dicetyl phosphate in 3 ml of dichloromethane, the solvent was removed using an evaporator to obtain a lipid mixture. Dissolve this in 3 ml of diethyl ether, and then add 1% enzyme solution (Enzyme-Amano, Protease A).
After adding 1 ml of solvent (3mM phosphate buffer pH 7.2) and mixing gently, the mixture was injected into a liposome preparation device and subjected to ultrasonic irradiation according to Experimental Example 1. The temperature is 25°
I went with C. The UF treatment was carried out in the same manner as in Experimental Example 1, and the enzyme encapsulation rate was determined. As a result, the retention efficiency was equivalent to that of the comparative example described below. The time required for preparation in the liposome preparation device was 13 minutes.

(Comparative Example) An example of a conventional liposome preparation method will be shown below. Diethyl ether was added to the lipid mixture obtained using the same ratio and method as in Experimental Example 1 to dissolve it, and the mixture was transferred to a medium-sized test tube, and then 5 ml of 1% enzyme solution was added and mixed by gentle shaking. This was irradiated with ultrasonic waves using a probe-type sonicator to form a W/O emulsion. The irradiation was performed at a temperature of 45° C. and a pulse irradiation of 150 W for 25 seconds. The sample was transferred to a 1000 ml eggplant-shaped flask, and the ether was distilled off under reduced pressure using an evaporator (400 mHg, 20°C, 5 minutes) to form a gel. After shaking for 15 minutes using a Vortex mixer, the remaining ether was removed using an evaporator again. (700 mmHg, 20°C, 1 hour) to obtain a paste-like REV. After diluting with a buffer to remove unencapsulated enzyme in the same manner as in Example 1, the enzyme retention efficiency in the liposome was determined to be 35.4%. Further, the actual preparation time from the ultrasonic treatment to the completion of REV was about 80 minutes, and the preparation time including the moving operation during this time was 90 minutes. In addition, the retention efficiency according to the comparative example is usually 28 to 3.
Although it is about 5%, the maximum value is shown. In addition, the comparison of preparation times was made in the direct part of forming REV. As described above, it can be seen that there is a significant difference between the comparative example according to the conventional method and the method using the apparatus of the present invention.

[0016]

Effects of the Invention According to the apparatus of the present invention, the preparation process is carried out in one sample container, which simplifies the operation compared to the conventional reverse phase evaporation method using beaker work. In addition, liposomes can be rapidly prepared and scale-up is easy. Conventional reversed-phase evaporation methods cannot be scaled up because they are carried out using an eggplant-shaped flask or the like.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of a liposome preparation device according to the present invention.

FIG. 2 is a liposome preparation flow according to the present invention.

[Explanation of symbols]

1 Ultrasonic converter 2 Jacket 3 Sample container 4 Chamber 5 Sample injection and discharge mechanism 6 Dilution water injection mechanism 7 Aspirator part 10 Conductivity measurement electrode 20 Sample tank 21 REV tank 22 Buffer tank

Claims (6)

[Claims] Claim 1: A sample container that also serves as the horn of an ultrasonic converter, a chamber that covers the sample container, a sample injection/discharge mechanism that supplies the sample to the sample container and collects prepared liposomes (hereinafter referred to as REV), and a sample container that A liposome preparation device comprising a dilution water injection mechanism for supplying dilution water and an aspirator section for reducing pressure inside a chamber. 2. The liposome preparation device according to claim 1, wherein the sample container is equipped with a temperature regulating jacket connected to a converter. [Claim 3] Claim 1, characterized in that the sample injection/discharge mechanism is configured such that the tip of a pipe that communicates with the sample tank and the REV tank through a pump and a switching valve faces the sample container. .2. The liposome preparation device according to any one of .2. 4. The dilution water injection mechanism according to claim 1, wherein the diluting water injection mechanism is configured such that the tip of a pipe having a valve and a pump communicating with the buffer tank faces the sample container. liposome preparation device. 5. The liposome preparation device according to claim 1, wherein the aspirator communicates with the upper part of the chamber through a mesh. [Claim 6] Lipid. The drug mixture is supplied to the sample container in the liposome preparation device according to claim 1 to emulsify it.
A liposome preparation method comprising forming an esicle in the same sample container, then diluting it and collecting REV from the sample container.